Tooth loss due to genetic causes, trauma, or decay constitutes a major health issue that results in substantial health care costs and burdens to patients. Since dental prostheses often result in resorption of the underlying bone, it is thought that a natural tooth is a more ideal replacement, and there is growing evidence that bioengineering dental tissues from patient-derived progenitor cells is a realistic goal. However, in order to safely use stem-cell based bioengineering approaches in clinical settings, much has to be learned about the processes that underlie the formation of dental tissues from stem cells. The continuously renewing mouse incisor provides a unique system for identifying the mechanisms that control formation and maturation of stem cell-derived dental cell types. Its unidirectional growth, with stem cell progeny at progressively increasing stages of maturity arrayed in a linear fashion, is of great advantage for studying stage-specific mechanisms. To identify cell type specific markers for the incisor stem cell system, I have recently built a gene co-expression network of the mouse incisor using an approach that clusters genes that co-vary in their expression across a large microarray data set generated from wild-type tissue samples. Co-variation often results from factors being implicated in the same biological process and is often driven by discrete cell types. Modules of co- expressed genes uncovered with this approach include modules specific to differentiated cell types such as ameloblasts and odontoblasts, modules that contain factors expressed in the label-retaining cell containing area, and several modules that appear to be specific to transit-amplifying (T-A) cells. This proposal aims to identify and test novel cell type specific markers with a focus on stem cell markers and markers for early stem cell progeny such as T-A cells. Cell type specificity will be assessed using multi-labeling expression studies and lineage tracing approaches and through evaluation in other renewing organs, markers will be further tested regarding their involvement in regulating stage specific processes during stem-cell based tooth renewal. The discovery of a co-expression module that contains splice-factors co-expressed in a domain specific to the transit-amplifying cells lead to the hypothesis that alternative splicing is crucial for T-A cell proliferaion. This will be tested by investigating the distribution of splice variants in stem cell progeny at sequential stages of maturation and by determining the consequences of disrupting the splicing machinery in the T-A cells using mouse genetics. Together, these studies will provide us with new biomarkers and greatly enhance our understanding of the cellular composition in the renewing mouse incisor as well as the stage-specific regulation of stem-cell based formation of dental tissues. Successful completion of these studies will provide us with important knowledge for advancing bioengineering strategies for generating dental tissues for clinical applications.